1. Field of the Invention
Many objects examined in the transmitted light microscope are highly transparent and colorless, and many objects examined with the reflected light microscope consist of substantially plane, mirror-like, surfaces on which the details of interest consists of very small topographical features having the same reflectivity as the surrounding surfaces.
Both of these kinds of objects are very difficult to examine because they neither absorb light nor deflect any significant amount of light out of the path of the objective. They therefore do not give rise to images showing significant local differences of intensity and hence are nearly or entirely invisible. The problem is most severe at high magnifications requiring objectives of large Numerical Aperture which accept light from a very wide angle.
2. Related Art
Many methods have been devised to render the detail in such objects clearly visible. All recognize that these types of objects produce phase changes in the light passing through or reflected from them. They are commonly referred to as phase objects. In transparent objects, phase changes are due to optical path differences resulting from local differences in thickness, refractive index, or both. In reflecting objects, phase changes are due principally to path differences caused by relative vertical displacements of different areas of the surface.
One class of microscopes devised to render phase objects visible is called Interference Microscopes. In these, light which has passed through or been reflected from an object is made to interfere with coherent light from the same source which has not passed through or been reflected from the object. Such microscopes tend to be complicated, expensive, difficult to use and give images which are difficult to interpret.
Another class which gives visible intensity contrast in images of phase objects is called Phase Contrast Microscopes. In these, light deflected by diffraction at the object is adjusted in phase with respect to the direct o undeflected light. The diffracted light is made to interfere with the direct light to give an intensity modulated image. Because the diffracted and direct light are not completely spacially separated, the Phase Contrast Microscope tends to produce annoying halos round large features, which can obscure nearby fine detail. However, such microscopes are widely used.
A more recent development is the Differential Interference Contrast Microscope, hereinafter, for convenience, referred to as a DIC microscope. Whilst the two classes of microscopes previously described render visible differences of phase in the object, the DIC microscope renders gradients of phase visible. Hence, the use of the word "differential" to indicate this sensitivity to the gradient or differential of phase. The background and areas of the object exhibiting uniform phase (whatever that phase might be) appear of the same intensity. Areas exhibiting phase gradients, such as edges, contours and concentration gradients, show up in lighter, darker or colored contrast against the background intensity. Visually the image gives the appearance of a three-dimensional object illuminated obliquely from one side. The observer can reverse the direction of apparent illumination and change from black and white to various colored contrast effects, at will.
To convert a regular transmitted light microscope to a DIC microscope, four components must be added: a polarizer, an analyzer and two prisms. The prisms are compound birefringent prisms of a general class known as Wollaston prisms, after their 19th century inventor. In a reflected light microscope, the objective also serves the function of a condenser. Hence, only one prism is required and the light passes through it twice.